Method of screening compounds which inhibit P. gingivalis lipopolysaccharide from inhibiting the extravasation of leukocytes

ABSTRACT

The lipopolysaccharide of bacteria associated with chronic inflammatory diseases is unable to induce expression of leukocyte adhesion molecules, or selectins, on endothelial cells, and is also capable of inhibiting the induction of selectin expression by bacteria normally associated with acute endotoxin disease. New approaches to treatment of these diseases, and the diagnosis of susceptibility to chronic bacterial-associated inflammatory diseases, are provided.

RELATED APPLICATION

This is a Division of application Ser. No. 08/337,614 filed Nov. 10,1994, which is a CIP of Ser. No. 08/150,635 filed Nov. 10, 1993 nowabandoned.

BACKGROUND OF THE INVENTION

This invention relates generally to methods for treating and preventingperiodontal diseases. Periodontal diseases are inflammatory disorders ofthe tissues supporting the teeth. These tissues are collectivelyreferred to as the periodontium and include the gingiva, periodontalligament, root cementum, and alveolar hone. Inflammation of theperiodontium is the major cause of tooth loss in the adult population inmost countries.

Periodontal diseases generally encompass two major and distinctsubclasses of disease, gingivitis and periodontitis. Gingivitis ischaracterized by inflammation of the gingiva without bone loss or lossof connective tissue attachment. Gingivitis is caused by bacterialaccumulation in the crevicular spaces. The gingiva becomes inflamedwithout spread to surrounding tooth support structures. Gingivitis maybe graded by severity, with mild gingivitis diagnosed clinically byerythema at the sites of inflammation. Moderate gingivitis involvesbleeding of the gingiva upon gentle probing, and severe gingivitis ischaracterized by a tendency for spontaneous gingival bleeding.Gingivitis is a precondition for, but does not necessarily lead to,periodontitis.

Periodontitis is an inflammatory disorder that can involve all tissuesof the periodontium. In periodontitis, oral bacteria accumulate at thejunction of the teeth and gingiva causing inflammation of the localperiodontal tissues. The inflammation degrades the collagen fibers ofthe periodontal tissues, causing loss of tooth support and theprogressive development of a space between the tooth and the gingiva(periodontal or gingival pocket). As the periodontitis progresses, theperiodontal pockets deepen, resulting in inadequate tooth support andtooth loss.

Many patients with severe periodontitis have serum antibodies toantigens of their infecting bacteria. The role these antibodies play inthe progression of periodontitis is not known, although many believethat they may be protective. Patients with high antibody titers haveless severe disease and fewer affected teeth than those with low titers.Gunsolley et al., J. Periodontol, 58:314-320 (1987) and Ranney et al.,J. Periodontol, 53:1-7 (1982). Serum antibodies in the presence ofcomplement significantly enhance phagocytosis and killing of periodontalpathogens by neutrophils. Underwood et al., J. Infect, Dis. (1993), andSjostrom et al., Infect. Immun. 62:145-151 (1994). Following periodontaltreatment, previously seronegative patients convert, and the capacity oftheir sera to stimulate phagocytosis and killing by neutrophilssignificantly increases along with increasing antibody titers andavidities. Chen et al., J. Periodontol. 62:781-791 (1991); Ou et al.,Prog. Abstr. Ann. Mtg. Intl. Assoc. Dent. Res. abstr. 2416 (1993).

Of the vast numbers of bacterial species which occupy the gingivalcrevice and the developing periodontal pocket, only a small group areconsidered putative pathogens. Prominent among the periodontopathicmicrobiota is Porphyromonas (Bacteroides) gingivalis. Porphyromonasgingivalis is a gram-negative anaerobic bacillus that has been stronglyimplicated as an etiologic agent in adult periodontal disease. Socranskyand Haffajee, J. Periodontol. 4:322 (1992). Recently, in a non-humanprimate model of periodontal disease, the emergence of this organismfrom the subgingival microbiota was associated with an increase inalveolar bone loss. Holt et al., Science 239:55 (1988). This data lendssupport to the hypothesis that a microbiological "bloom" of P.gingivalis may be associated with progression of the disease. Socranskyand Haffajee, J. Clin. Periodontol, 13:617 (1986). The relationshipbetween the presence of P. gingivalis and the chronic inflammatorynature of the disease remains unclear.

One of the first steps in the inflammatory process is the emigration ofleukocytes from the vascular compartment to extravascular tissues.Lasky, Science 258:964 (1992). Leukocyte emigration is initiated by aninflammatory stimulus which induces the expression of selectin moleculeson the surface of vascular endothelial cells. Potent inducers ofselectin include E. coli lipopolysaccharide (LPS), tumor necrosis factor(TNF) and interleukin-1 (IL-1). An initial binding of low affinity andhigh avidity occurs between carbohydrate ligands on the leukocytes(e.g., the sialyl Lewis^(x) molecule) and the selectin molecules on thevascular endothelium. This low affinity binding results in theleukocytes "rolling" along the endothelial wall in a manner that permitsa more stable, higher affinity binding to develop via the leukocytesintegrin molecules and the ICAM receptors expressed on the surfaces ofthe endothelial cells. The expression of both P- and E-selectin has beenshown to be transient in vitro and is believed to be transient in vivo.This is consistent with evidence that suggests continued expression ofthese molecules could result in inflammatory disease due to thecontinued evasiation of leukocytes from vascular to tissue compartments.

However, normal trafficking of leukocytes from the vascular compartmentto gingival tissues is clearly required for the prevention ofperiodontal disease. Anderson et al., J. Infect. Dis. 152:668 (1985);Etzoni et al., N. Engl. J. Med. 327:1789 (1992). Leukocytes frompatients with congenital defects in the expression of the leukocyte B2integrin receptor CD11/CD18 are unable to bind to their respective ICAMreceptors on endothelial cells, and the patients typically suffer fromsevere periodontal disease. Anderson et al., supra. Recently, acongenital leukocyte adhesion deficiency in the expression of thesialyl-lewis X ligand for E-selectin has been described. Etzoni et al.,supra. This defect resulted in severe periodontal disease, therebyconfirming the requirement for a functional selectin pathway for theprevention of periodontal disease.

Traditional microbiological and immunological approaches to controllingperiodontal disease have attempted to eliminate the pathogenicmicroorganisms or maintain them at very low levels. These efforts havefocused on antibiotic treatment and, more recently, the generation ofprotective immune responses by vaccination against bacterial antigens.In one study non-human primates immunized with formalin-fixed P.Gingivalis demonstrated significant increases in serum antibody titersto the organism and a significant reduction in alveolar bonedestruction. Perrson et al., Infect, Immun., 62:1026-1031 (1994). Eventhough the vaccine was effective in suppressing or arresting bone lossand lessening attachment loss, the subgingival plaque in immunizedanimals still harbored very large numbers of P. gingivalis, suggestingthat protection against tissue destruction may be multifactorial.

In an earlier study, Ebersole et al. (Infect, Immun. 59:3351-3359(1991)) immunized nonhuman primates with P. gingivalis and Prevotellaintermedia. They showed that active immunization could elicit a systemicimmune response against the organisms, and that P. gingivalisimmunization could significantly inhibit the emergence of the speciesduring subsequent disease progression. Subgingival plaque indices,however, indicated that few changes could be attributed to immunization,and both bleeding on probing and loss of attachment were higher in areasof ligature-induced periodontitis in immunized animals than inplacebo-treated controls. In fact, a significant increase in bonedensity loss was observed in the ligated teeth of immunized animals.Ebersole et al. noted that it is possible that immunization with one oreven several microorganisms in a complex ecosystem could exacerbate thedestructive events of such a multifactorial disease.

Despite an understanding of the microbial origins of periodontal diseaseand its temporal progression, means to arrest or even eliminate thedisease have eluded investigators. Quite surprisingly, the presentinvention provides a means to interfere with and modulate the temporalprogression of this and other chronic inflammatory diseases associatedwith gram-negative bacterial infections, and further fulfills otherrelated needs.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions to treat andprevent inflammatory disease states, particularly chronic inflammatorydiseases, that are associated with gram-negative bacterial infection.The most prevalent of these diseases are those associated with anaerobicgram-negative organisms, such as periodontal disease, i.e.,periodontitis and gingivitis, and ulcers. The invention provides amethod for modulating the progression of periodontal disease in a mammalby administering a compound which inhibits the ability of P. gingivalisto inhibit the extravasation of leukocytes from the vascular endotheliumto gingival tissues.

The compound useful in the present methods can be specific for LPS ofthe organism or specific to the endothelial cell ligand which binds theLPS molecule, e.g., polyclonal or monoclonal antibody, or can be acomposition of compounds which recognize the components of theLPS-ligand interaction and thereby inhibit the immunosuppressivedown-regulation of selectin expression. In the treatment or preventionof periodontal disease, typically the compound is administered to theperiodontium, by mouthwash, aerosol, paste or salve. The compound ormixture thereof is administered in an amount sufficient to inhibit theability of P. gingivalis lipopolysaccharide to inhibit the extravasationof leukocytes from the vascular endothelium to gingival or otherafflicted tissues. In one embodiment the compounds are monoclonalantibodies that bind specifically to P. gingivalis lipopolysaccharide,and in another the compound is an enzyme that specifically degradescomponents of the lipopolysaccharide of P. gingivalis or other causativeorganism as described herein, e.g. H. pylori. The compounds useful inthe present methods can be targeted to the affected tissues, e.g.,periodontium or gingival tissues, by an antibody that binds to P- orE-selectin, where the antibody can be a bifunctional antibody capable ofbinding to both P- and E-selectin, and in some embodiments the compoundis directly linked to the antibody.

In other embodiments the invention provides methods for screeningcompounds which attenuate the ability of LPS that inhibits theextravasation of leukocytes from the vascular endothelium. The methodscomprise contacting cells which are capable of expressing a selectinmolecule, such as HUVECs, with LPS from a selected organism, such as P.gingivalis, in the presence and absence of the compound being screenedfor the ability to inhibit LPS-induced inhibition of selectinexpression. Expression of selectin is stimulated, e.g., by exposure toE. coli LPS, tumor necrosis factor, or interleukin-1, and the expressionof selectin in the presence or absence of said compound is measured andthe ability of the compound to attenuate or prevent LPS-inducedinhibition of selectin expression determined. This method isparticularly useful for screening mutants of AOAH which have increasedability to inhibit or degrade the LPS molecule being tested.

The invention also provides a method for diagnosing host susceptibilityto chronic inflammatory disease associated with an anaerobic ormicroaerophilic gram-negative bacterial infection, such as periodontaldisease, chronic gastritis or gastroduodenal ulcers. The methodcomprises contacting cells of said host capable of expressing selectins,such as endothelial cells, with a diagnostic marker specific for theligand that binds to LPS of the disease-associated bacteria, e.g., P.gingivalis or Helicobacter pylori. The presence of said ligand isindicative of the susceptibility of the host to LPS-mediated inhibitionof selectin expression and thus the chronic inflammatory disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the stimulation of E-selectin expression with varyingconcentrations of E. coli (ATCC 29552) and the relative absence ofE-selectin expression by P. gingivalis (ATCC 33277).

FIG. 2 illustrates the results of E-selectin expression using wholebacteria; E. coli ATCC 29552, P. gingivalis ATCC 33277, and H. pyloriATCC 43504 were examined in five separate experiments. P. aeruginosaATCC 27313 was examined in two separate experiments. The mean andinterassay standard deviation from the mean are shown.

FIG. 3 shows the stimulation of E-selectin expression with LPSpreparations. Each assay was performed on at least four separateoccasions. The mean and inter-assay standard deviation from the mean areshown.

FIG. 4 shows the stimulation of E-selectin expression with bacterialcell wall preparations. Each experiment was performed on at least threeseparate occasions with similar results. The data are presented as theaverage of a typical experiment performed in triplicate.

FIG. 5 depicts the effects of LPS preparations from E. coli, variousstrains of P. gingivalis, and B. forsythus on E-selectin expression byhuman umbilical vascular endothelial cells.

FIG. 6A shows the ability of P. gingivalis LPS preparations to inhibitE-selectin expression induced by varying concentrations of E. coli LPS.

FIG. 6B shows the effect of P. gingivalis LPS on E-selectin expressionthat is induced by tumor necrosis factor (TNF).

FIG. 7 illustrates the inhibition of E selectin expression induced by E.coli LPS by the LPS of P. gingivalis compared to a relative lack ofinhibition by the lipid A or polysaccharide ("LPS-PS") fractions of P.gingivalis LPS.

FIG. 8 demonstrates the ability of various dilution of rabbit antiseraprepared against P. gingivalis to block the ability of P. gingivalis(ATCC 33277) LPS to inhibit E. coli LPS-mediated upregulation ofE-selectin on HUVECs.

FIG. 9 shows neutrophil adherence to HUVEC treated with different LPSpreparations. At maximum binding (2000 units) approximately 50% of theneutrophils were bound. Three separate experiments were performed. Themean and inter-assay standard deviation from the mean are shown.

FIG. 10 shows that increasing concentrations of P. gingivalis LPSinhibits monocyte chemoattractant protein 1 (MCP-1) RNA expression thatis stimulated in human gingival fibroblasts by E. coli LPS.

FIG. 11 shows that E. coli LPS was able to induce K light chainexpression but P. gingivalis LPS did not in a murine B lymphoma line, 70Z/3. P. gingivalis LPS was able to inhibit the ability of the E. coliLPS to induce K light chain expression.

FIG. 12 demonstrates the effect of monoclonal antibody to P. gingivalisLPS on E. coli induced neutrophil adhesion.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides methods and compositions to treat andprevent chronic inflammatory disease states that are associated withanaerobic gram-negative bacterial infection. The most prevalent of thesediseases are periodontal disease, i.e., periodontitis and gingivitis,and ulcers. As with most gram-negative organisms, the bacteriaassociated with these diseases have lipopolysaccharide ("LPS") in theouter bacterial membrane. Among members of Enterobacteriaceae LPS is apotent inflammatory mediator and can even lead to endotoxin shock. Ithas been a paradox that the LPS of gram-negative anaerobes involved inchronic inflammatory diseases are generally less toxic.

As one aspect of the present invention, it has now been discovered thatLPS from gram-negative anaerobes can be a potent inhibitor of earlyaspects of the normal inflammatory process, i.e., the emigration ofleukocytes from the vascular endothelium. Inhibition or disruption ofthe normal inflammatory pathway from the vascular endothelium by thegram-negative anaerobic bacteria represents a potential new mechanism ofhost invasion.

Although inhibition of leukocyte emigration is not complete, sinceperiodontitis, gingivitis and the like are replete with neutrophils andlymphocytes, the present invention demonstrates that certain bacterialike P. gingivalis have adapted a host evasion strategy that involves aform of inflammation masking. By containing a less virulent andimmunosuppressive LPS they have changed the very molecule which wouldnormally lead to their detection and elimination by the immune system.The present invention provides methods and compositions for preventingor counteracting the inhibition of the normal immune function by theseinfections, for restoring immune function at sites of harmfulbacterial-induced inflammation, and methods for diagnosing hostsusceptibility to bacterial-induced inflammatory responses that arepotentially destructive of host tissue.

The present invention provides the ability to inhibit the disruption ofa normal inflammatory response that is mediated by the LPS of certaingram-negative bacteria, particularly the gram-negative anaerobicbacteria associated with chronic diseases in mammalian hosts, includinghuman hosts. LPS has been shown to inhibit expression of selectinmolecules on the surfaces of vascular endothelial cells, therebyimpeding the normal flow of leukocytes, particularly neutrophils, to theextravascular tissue and the site of infection. Adequate responses byneutrophils and other leukocytes are essential to preventing orovercoming a bacterial infection. By reversing or preventing thesuppression of emigration of these leukocytes through the vascularendothelium according to the present invention, thereby permitting amore "normal" immune response, the gram-negative infections can be morereadily treated by conventional therapies, as desired. The genesencoding the selectin cell surface glycoproteins, including E-selectin(ELAM) and P-selectin (GMP140/PADGEM, have been cloned and sequenced.See, e.g., Bevilacqua et al., Science 243:1160 (1989) and Johnston etal., Cell 56:1033 (1989), respectively, which are incorporated herein byreference.

The present invention is directed toward prevention and treatment of awide variety of infections due to anaerobic gram-negative bacilli thatare typically associated with pathological inflammatory diseases. One ofthe most prevalent of such infections is associated with periodontaldisease, particularly gingivitis and periodontitis. These diseases aretypically associated with polymicrobial infections, but prominent amongthe microbiota associated with these diseases are members of the generaBacteroides (e.g., B. melaninogenicus), Porphyromonas (e.g., P.gingivalis, P. intermedia), Prevotella (e.g., P. denticola, P.loescheii), Eikenella (e.g., E. corrodens), and Wolinella (e.g., W.recta).

Other pathological inflammatory diseases associated with infections dueto microaerophilic gram-negative bacilli and that are susceptible totreatment or prevention according to the present invention includechronic gastritis or gastroduodenal ulcers, which have recently beenassociated with chronic infection by Helicobacter pylori.

In another embodiment the invention concerns the treatment of infectionsdue to organisms of the family Pseudomonadaceae. These gram-negativebacteria typically cause infections in the presence of immunosuppressiveconditions in a host, and are extremely difficult to treat withconventional antibiotic therapy. The LPS from these organisms inhibitsthe expression of selectins by vascular endothelial cells, therebyrendering the bacteria less exposed to normal host defenses. Preventingor attenuating the relative absence of selectin expression according tothe present invention permits a more normal and effective host immuneresponse, either separately or in conjunction with other treatmentmodalities. Clinically normal periodontal tissue has been reported tohave elevated levels of expression of E-selectin and the inflammatorychemokine MCP-1. The increased expression of these inflammatorymediators, in close proximity to bacterial plaque, is consistent with astate of low level inflammation in clinically normal tissue. Offered byway of possible explanation but not limitation, the ability of P.gingivalis LPS to block direct E-selectin expression in a localenvironment such as the periodontium may contribute to the colonizationof the tooth root surface and result in the bacterial blooms that occurin periodontal disease. In addition, inhibition of the inflammationnormally induced by a wide variety of other bacteria may contribute tothe characteristically large numbers of different bacteria found inthese lesions.

In accordance with the present invention, compounds which can inhibitthe interaction of the immunosuppressive LPS with its correspondingligand on the endothelial cell are effective in permitting a moreeffective immune response, i.e., the emigration of leukocytes, andparticularly neutrophils, to the extravascular tissue where theinfecting organisms can be attacked and destroyed. Compounds which areeffective inhibitors of the anaerobic or microaerophilic bacterial LPSinteraction with the ligands on the endothelial cells are identified inscreening assays and the like.

Particularly useful inhibitors of the LPS-ligand interaction, and thuseffective mediators of the chronic inflammatory disease process, areantibodies and binding fragments thereof specific for the LPS or thecorresponding ligand. Thus, the antibodies or other compounds which areemployed in the compositions and treatments of the present invention arethose which demonstrate the ability to inhibit or reverse the bacterialLPS-mediated inhibition of selectin expression, e.g., the inhibition ofE. coli LPS-induced selectin stimulation as demonstrated for P.gingivalis LPS in the Examples below.

Thus, the antibodies and binding fragments thereof useful in the presentinvention can be either polyclonal or monoclonal, but preferably aremonoclonal. If polyclonal, they can be in the form of antiserum ormonospecific antibodies, such as purified antiserum which has beenproduced by immunizing animals with P. gingivalis or the purified LPSthereof. Preferably, however, the antibodies are monoclonal antibodiesso as to minimize the administration of extraneous proteins to anindividual. Monoclonal antibodies which bind to the different componentsof the anaerobic or microaerophilic gram-negative bacterial LPS moleculeor the endothelial cell ligands thereof can be prepared according towell known protocols. See. e.g., Skare et al., J. Biol. Chem.268:16302-16308 (1993), U.S. Pat. Nos. 4,918,163 and 5,057,598, whichare incorporated herein by reference.

For administration to humans, e.g., as a component of a composition forin vivo treatment, the monoclonal antibodies are preferablysubstantially human to minimize immunogenicity, and are in substantiallypure form. By "substantially human" is meant that the immunoglobulinportion of the composition generally contains at least about 70% humanantibody sequence, preferably at least about 80% human, and mostpreferably at least about 90-95% or more of a human antibody sequence.When referring to "antibody," it will be understood thatnon-immunoglobulin sequences may optionally be present in the moleculeso long as the molecule retains the ability to bind the LPS or LPSligand present on the endothelial cell.

As the generation of human monoclonal antibodies to a ligand present onhuman cells may be difficult with conventional human monoclonal antibodytechniques, it may be desirable to transfer antigen binding regions(e.g. the F(ab')₂, variable or hypervariable (complementaritydetermining) regions), of non-human monoclonal antibodies, such as froma murine monoclonal antibody that has been made to ligand purified fromcells via an affinity interaction with the LPS, to human constantregions (Fc) or framework regions using recombinant DNA techniques,thereby producing substantially human molecules. Such methods aregenerally known in the art and are described in, for example, U.S. Pat.No. 4,816,397, EP publications 173,494 and 239,400, which areincorporated herein by reference. Alternatively, one may isolate DNAsequences which code for a human monoclonal antibody or portion thereofthat specifically binds to the human ligand, or to anaerobic ormicroaerophilic bacterial LPS antigen by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989), and described in WO 90/14430, incorporatedherein by reference, and then cloning and amplifying the sequences whichencode the antibody (or binding fragment) of the desired specificity. Inyet other embodiments, single chain binding polypeptides can be madewhich bind to the immunosuppressive anaerobic or microaerophilicbacterial LPS or to the corresponding cellular ligand(s) thereof. Thesesingle chain polypeptides may be produced by cloning and Joining thevariable regions of the heavy and light chains of a monoclonal antibodywhich binds to the LPS antigen or endothelial cell ligand(s) thereof.Methods for the production of single chain binding polypeptides aredescribed in detail in, e.g., U.S. Pat. No. 4,946,778, which isincorporated herein by reference.

Other compounds which are capable of binding to the immunosuppressiveanaerobic or microaerophilic bacterial LPS molecule or to the cellularligand thereof and which inhibit the immunosuppressive effects of thecorresponding bacteria, but which are not derived from immunoglobulinmolecules, can be isolated according to established protocols. Forexample, LPS-specific or LPS/ligand-specific binding polypeptides can beisolated by screening vast libraries of random or semi-randompolypeptides. The polypeptide libraries can be expressed and isolated asa component of a phage coat protein (e.g., Scott and Smith, Science249:386 (1990); Dower et al., WO 91/19818), as part of a polyribosome(e.g., Kawasaki, WO 91/05058), or without a ribosome present (Gold etal., WO 93/03172), each of which publications is incorporated byreference herein. Once the binding molecule is identified according tothe desired selection procedure, the molecule is tested as describedherein for the ability to inhibit the suppression (e.g., by P.gingivalis) of the upregulation of selectin expression as can be causedby, e.g., E. coli LPS, TNF or IL-1, and thus are useful in treating orpreventing the chronic inflammatory process associated with suchbacteria. Once the monoclonal antibody, LPS- or ligand-bindingpolypeptide is identified it can be expressed in large quantities forproduction purposes.

The methods of the invention can also be used in a screening assay toidentify effective compounds. According to one protocol, the compoundsare screened for the ability to ameliorate P. gingivalis or H. pyloriLPS-induced inhibition of selectin expression. Cells which are capableof expressing a selectin molecule, e.g., E- or P- selectin, arecontacted with the bacterial LPS, e.g., that of P. gingivalis, in thepresence and absence of the compound being screened for the ability toinhibit P. gingivalis LPS-induced inhibition of selectin expression.Selectin expression is stimulated and measured in the presence orabsence of the compound being tested. The ability of the compound toinhibit P. gingivalis LPS-induced inhibition of selectin expression isthen determined. The cells expressing selectin are conveniently humanumbilical endothelial cells (HUVECs), and selection expression ispreferably induced by E. coli LPS, but tumor necrosis factor,interleukin-1 or other stimulators may also be tested.

Compounds which bind the immunosuppressive anaerobic or microaerophilicbacterial LPS molecule or to the cellular ligand thereof and whichinhibit the immunosuppressive effects of the corresponding bacteria,such as monoclonal antibodies, are useful in a wide variety oftherapeutic and prophylactic settings. These compounds are administeredin compositions to prevent and/or treat the chronic inflammatorydiseases associated with infections by such bacteria, e.g.,periodontitis, gingivitis, chronic gastritis or gastroduodenal ulcers,and the like.

Neutrophils, monocytes and vascular endothelial cells have been shown tocontain acyloxyacyl hydrolase ("AOAH"), an enzyme that detoxifiesbacterial LPS. The presence of neutrophils that contain AOAH, or thedelivery of purified AOAH to the infected tissue, may facilitate a morerapid resolution of the infection. The isolation and purification ofAOAH from human neutrophils has been described, e.g., U.S. Pat. No.5,013,661, incorporated herein by reference, as has the cloning andexpression of AOAH molecules and subunits thereof by recombinant DNAtechniques, PCT publication WO 92/04444 and U.S. Pat. No. 5,281,520,incorporated herein by reference. AOAH can be targeted to the sites ofchronic bacterial infection via antibodies which bind to selectins,e.g., E-selectin or P-selectin. The targeting antibodies are preferablymonoclonal antibodies, and can be linked directly to the AOAH molecule,as a fusion protein, or indirectly, e.g., contained within a liposomepreparation targeted by the anti-selectin antibody or binding fragmentthereof. The AOAH can be linked to an antibody which has bifunctionalspecificity, i.e., capable of binding to both E-selectin and P-selectin.

In other instances, the immunosuppressive yet relatively nontoxic LPSprepared from these organisms (e.g., P. gingivalis) can also beadministered in sufficient quantities to treat or at least amelioratethe endotoxin shock that is often associated with acute infections bygram-negative organisms having more toxic LPS moieties, e.g., E. coli,Enterobacter, Salmonella, and the like. The therapeutically administeredLPS can be modified to further reduce the toxicity thereof in a patientwhile retaining the ability to inhibit the stimulation of selectinexpression by the LPS of the infecting organism.

The LPS molecules, or mimetics thereof, that inhibit the expression ofselectin molecules can also be used as drugs to inhibit otherselectin-mediated inflammation, such as that involved in accumulation oflymphocytes in the skin in certain skin diseases, e.g., psoriasis andcontact dermatitis, during acute inflammation of the lung (adultrespiratory distress syndrome), reperfusion injury, and the like.

As used herein, the terms "treatment" or "treating" include: (1)preventing such disease from occurring in a subject who may bepredisposed to these diseases but who has not yet been diagnosed ashaving them; (2) inhibiting these diseases, i.e., arresting theirdevelopment; or (3) ameliorating or relieving the symptoms of thesediseases, i.e., causing regression of the disease states. For example,with respect to chronic bacterial infections associated with thesediseases, treatment according to the present invention will increase thenumber of neutrophils at the site of infection and thereby increase thephagocytosis or destruction of the infecting bacteria or their toxiccomponents.

The monoclonal antibodies or other compounds useful in the presentinvention can be incorporated as components of pharmaceuticalcompositions containing a therapeutic or prophylactic amount of at leastone of the monoclonal antibodies or binding fragment thereof with apharmaceutically effective carrier. For example, monoclonal antibodiesor binding fragments thereof to the LPS can be combined with differentantibodies which bind to different epitopes on the LPS molecule, or toepitopes on the LPS ligand on the cellular surface, or to other cellularreceptors such as E- or P-selectin, to form a treatment "cocktail."

In preparing the pharmaceutical compositions useful in the presentmethods, a pharmaceutical carrier should be employed which is anycompatible, nontoxic substance suitable to deliver the antibodies orbinding fragments thereof or therapeutic compounds identified inaccordance with the methods disclosed herein to the patient. Sterilewater, alcohol, fats, waxes, inert solids and even liposomes may be usedas the carrier. Pharmaceutically acceptable adjuvants (buffering agents,dispersing agents) may also be incorporated into the pharmaceuticalcomposition. The antibodies and pharmaceutical compositions thereof areparticularly useful for parenteral administration, i.e., intravenously,intraarterially, intramuscularly, or subcutaneously. Localadministration can also be effective, particularly in the treatment orprevention of periodontal disease, i.e., periodontitis and gingivitis,where the compound is contained in a mouthwash solution, paste, salve,ointment or gel and is applied directly to the affected tissues. Theconcentration of compound such as an antibody in a formulation foradministration can vary widely, i.e., from less than about 0.5%, usuallyat least 1% to as much as 15 or 20% or more by weight, and will beselected primarily based on fluid volumes, viscosities, etc., preferredfor the particular mode of administration selected. Actual methods forpreparing administrable compositions will be known or apparent to thoseskilled in the art and are described in more detail in, for example,Remington's Pharmaceutical Science, 17th Ed., Mack Publishing Co.,Easton, Pa. (1985), which is incorporated herein by reference.

The compounds of the invention useful in inhibiting theimmunosuppression associated with the LPS of gram-negative anaerobic ormicroaerophilic bacteria can be administered for prophylactic ortherapeutic treatment. In treatments intended for prophylacticapplications, the compositions are administered to a patient susceptibleto periodontal disease or other chronic bacterial-induced inflammatorydisease, such as chronic gastritis or gastroduodenal ulcers, forexample. To prevent recurrent disease and the sequelae thereof, thecompositions may be administered daily, weekly or other scheduledmaintenance therapy. The regimen will also depend on the dosage andeffectiveness thereof, the intended use and the patient's general stateof health. The treating physician or dentist will select dose levels andpattern of administration, i.e., route and single or multipleadministrations.

In therapeutic applications, the compounds of the invention useful ininhibiting the immunosuppression associated with the LPS ofgram-negative anaerobic or microaerophilic bacteria are administered toa patient already suffering from periodontitis or gingivitis or otherchronic bacterial-induced inflammatory disease, such as chronicgastritis or gastroduodenal ulcers, in an amount sufficient to at leastpartially arrest the infecting and, hence, inflammatory process. Anamount adequate to accomplish this is defined as a "therapeuticallyeffective dose." Amounts effective for this use will depend upon thecompound being employed, the route of administration, the severity ofthe disease and the general state of the patient's health. Determinationof an effective amount of a compound of the invention to inhibit theimmunosuppressive components of the infecting bacteria can be determinedthrough standard empirical methods which are well known in the art.Reversal of inhibition of selectin stimulation or merely stimulation ofselectin expression, emigration of neutrophils and other leukocytes, andthus efficacy of the subject compositions, can be monitored with avariety of well known in vitro diagnostic procedures.

The invention also provides a method for diagnosing host susceptibilityto chronic inflammatory disease associated with an anaerobic ormicroaerophilic gram-negative bacterial infection, such as periodontaldisease, chronic gastritis or gastroduodenal ulcers. The methodcomprises contacting cells of said host capable of expressing selectins,such as endothelial cells, with a diagnostic marker specific for theligand that binds to LPS of the disease-associated bacteria, e.g., P.gingivalis or Helicobacter pylori. The presence of said ligand isindicative of the susceptibility of the host to LPS-mediated inhibitionof selectin expression and thus the chronic inflammatory disease.

The following examples are offered by way of illustration of theinvention, not by way of limitation.

EXAMPLE I

In this Example the ability of P. gingivalis, an important periodontalpathogen, to stimulate expression of E-selectin, a key initial componentof the inflammatory pathway, was examined. An understanding into therelationship between these components is necessary to further understandand treat the disease. Quite unexpectedly, P. gingivalis failed tostimulate E-selectin expression.

Initially the ability of whole bacteria to stimulate E-selectinexpression on human umbilical cord endothelial cells (HUVEC) wasexamined. E-selectin expression was stimulated with varyingconcentrations of E. coli ATCC 29552 and P. gingivalis ATCC 33277. E.coli ATCC 29552, which contains the 0111:B4 serotype LPS, was obtainedfrom the ATCC; P. gingivalis strains were obtained from Dr. AaronWeinberg of the University of Washington Department of Periodontics,Seattle, Wash.

Several P. gingivalis strains were examined in addition to ATCC 33277,including 381, A7A1-28, A7436, and 5083. Bacteria were grown on BrucellaBlood (Difco) agar supplemented with vitamin K and hemin as described(Nash et al., Manual Clinical Microbiology, chap. 121, 1226 (Amer. Soc.Microbiol.) Wash. D.C. (1991)). Cultures were incubated for 72 hours at37° C. under either microaerophilic, anaerobic or aerobic conditions asappropriate. Cultures were aseptically suspended in Media 199supplemented with 4 mM L-glutamine, 90 μg/ml heparin, 1 mM sodiumpyruvate, 1 mg/ml human serum albumin, and diluted in the same media tothe indicated cell number by calculation from a predetermined conversionfactor. Bacterial suspensions were added to a monolayer of fourthpassage HUVEC plated in a fibronectin precoated 96 well plate (Costar,flat-bottom) as with the addition of 5% pooled human serum (GeminiBioproducts). After four hours incubation the plates were washed andassayed for the presence of E-selectin. Each assay was performed induplicate on three separate occasions. The results of a typical assayare shown in FIG. 1. Endothelial cell viability was determined onduplicate plates after the four hour incubation by the calcein method,as described by the manufacturer (Live/Dead™ Viability/CytotoxicityAssay, Publ. MP85, Molecular Probes, Inc. 1991).

As shown in FIG. 1, E. coli whole cells were a potent inducer ofE-selectin expression. In contrast to E. coli, the addition of P.gingivalis to the endothelial cells did not result in the expression ofE-selectin. All strains of P. gingivalis examined failed to stimulateE-selectin expression. Strains examined included the monkey strain(5083) previously used to demonstrate that P. gingivalis can function asa primary pathogen in periodontal disease, as well as a strain (A7436)found to be particularly virulent in a rodent model of infection.

Microscopic examination of the endothelial cell layer after bacterialstimulation revealed no change in endothelial cell shape or loss in cellnumber. A more quantitative estimate of endothelial cell viability wasdetermined by measuring the hydrolysis of calcein-AM. This reagentdetects hydrolysis mediated by an esterase present in eukaryotic but notbacterial cells. The lack of E-selectin expression could not beattributed to endothelial cell toxicity since even high concentrationsof these bacteria were not toxic as assayed by these parameters.

These results demonstrated that P. gingivalis was unable to stimulateE-selectin even though this organism has been clearly associated withthe inflammatory lesions found in periodontal diseases.

EXAMPLE II

Similar to Example I, this Example examines the comparative abilities ofP. gingivalis, E, coli, S. typhimurium, P. aeruginosa, and H. pylori tostimulate expression of E-selectin.

HUVECs (Clonetics, San Diego, Calif.) were maintained in HUVEC growthmedia Media-199 (Gibco, Gaithersberg, Md.) containing 4 mM L-glutamine,90 μg/ml heparin, 1 mM Na pyruvate, 30 μg/ml endothelial cell growthstimulant (Biomedical Products, Bedford, Mass.) and 20% fetal bovineserum (Hyclone Lab, Logan, Utah). Cells were used at the fourth passage.Initial experiments conducted on cells in the second or third passageshowed no apparent difference in the E-selectin response. HUVEC (1.4×10⁴/well) were plated in a fibronectin precoated 96 well flat-bottom plate(Costar, Pleasonton, Calif.) in M-199 growth medium the day beforestimulation by bacterial cells or bacterial cell products.

Bacteria used included E. coli ATCC 29552, JM 83, MC 1061, and MC 4100(Dr. J. Somerville, Bristol-Myers Squibb), P. gingivalis strains ATCC33277, strain 381 A7A1-28, A7436, and 5083, P. aeruginosa strain ATCC27313, a Bacteroides forsythis strain, and H. pylori ATCC 43504.

For the human E-selectin expression assay, on the day of the assay,bacterial cultures were suspended (from plate grown cells) in M-199stimulation medium (Media-199 containing 4 mM L-glutamine, 90 μg/mlheparin, 1 mM Na pyruvate, 1 mg/ml human serum albumin, and 5% poolednormal human serum (Gemini Bioproducts, Calabasas, Calif.)) and dilutedin the same media to the desired cell number by calculation from apredetermined conversion factor. Conversion factors for each bacterialstrain were determined by plate count analysis performed in triplicateby standard procedures for bacterial enumeration. HUVEC were washed withM-199 stimulation without serum, bacterial preparations were then addedto the HUVEC monolayer and incubated for four hours at 37° C. under 5%CO₂. After the stimulation interval, media was removed, the cells werewashed twice in cold PBS, fixed with 0.5% glutaraldehyde (in cold PBS)and placed at 4° C. for 10 minutes. Cells were washed four times withPBS containing 3% pooled goat serum (Sigma, St. Louis, Mo.) and 0.02MEDTA (blocking buffer). After the last wash 0.2-0.3 ml of blockingbuffer was added to each well and the plates were stored overnight at 4°C. (this blocking step was complete after one hour, but for convenienceovernight incubations were routinely employed). Blocking buffer wasremoved and 0.1 ml of anti-E-selectin monoclonal antibody (R and DSystems, Minneapolis, Minn.) at 0.25 μg/ml in blocking buffer was addedto each well and the plate was incubated at 37° C. for 1 hour. Plateswere washed four times in blocking buffer and 0.1 ml of F(ab')2 goatanti-mouse IgG specific HRP conjugated second step antibody (JacksonImmunoresearch Labs, West Grove, Pa.) diluted in blocking buffer wasadded to each well. Plates were incubated at 37° C. for 1 hour, washedfour times with blocking buffer, and 0.1 ml of chromogen reagent (TMB insubstrate buffer, Genetic Systems, Redmond, Wash.) was added. Thereaction was stopped with 0.1 mL of 1N H₂ SO₄ per well and the platesread in an ELISA reader (BioTek Instruments, Winooski, Vt.) at 450/630nm. Endothelial cell viability was determined on duplicate plates afterthe four hour incubation by the calcein-AM method.

The results for the ability of whole bacteria to stimulate E-selectinexpression on HUVEC are shown in FIG. 2. E. coli cells were a potentinducer of E-selectin expression. H. pylori and P. gingivalis were verypoor stimulators of E-selectin expression. At the highest concentrationsof bacteria added to the assay, only low levels of E-selectin wereobserved. Similar to E. coli, P. aeruginosa induced nearly maximallevels of E-selectin expression in the assay, however, approximatelythree more logs of bacteria were required. The degree of E-selectinstimulation was consistent among different species in a single genus.For example, several different strains of E. coli ATCC 25922 and S.typhimurium (Table 1) displayed similar dose response curves as shownfor E. coli ATCC 29552. Five different strains of P. gingivalis wereexamined and failed to stimulate E-selectin expression. Strainsexamined, but not shown in FIG. 2, included a monkey strain (5083) usedto demonstrate that P. gingivalis can function as a primary pathogen inperiodontal disease, a strain (A7436) that is particularly virulent in arodent model of infection, and two other strains designated A7A1-28 and381. Two different strains of P. aeruginosa yielded a similar doseresponse curve (FIG. 2 and Table 1). Microscopic examination of theendothelial cell layer after bacterial stimulation revealed no change inendothelial cell shape or loss in cell number at the bacterialconcentrations employed in the assay.

                  TABLE 1                                                         ______________________________________                                        E-selectin stimulation: Effect of different strains of bacteria*              Bacteria                                                                             E. coli    E. coli S. typhimurium                                                                         P. aeruginosa                              (cfu)  (ATCC 25922)                                                                             (JM83)  (A568)   (ATCC 27316)                               ______________________________________                                        10.sup.9                                                                             ND         ND      ND       .84 ± .01                               10.sup.8                                                                             ND         ND      ND       .36 .05                                    10.sup.7                                                                             ND         ND      ND       .07 .05                                    10.sup.6                                                                             .71 .20    .83 .07 .66 .14  0                                          10.sup.5                                                                             .71 .06    .74 .05 .44 .22  0                                          10.sup.4                                                                             .52 .08    .38 .11 .21 .04  0                                          10.sup.3                                                                             .20 .10    .19 .06 ND                                                  10.sup.2                                                                             .03 .04    ND      ND                                                  ______________________________________                                         *Values represent the mean and standard deviation from at least three         separate experiments for each strain examined. E. coli ATCC 25922 is a        clinical isolate whereas E. coli JM83 is a laboratory strain. Other           laboratory strains (MC1061 and MC4100) yielded similar results in two         separate experiments.                                                    

A more quantitative estimate of endothelial cell viability wasdetermined by measuring the hydrolysis of calcein-AM. This reagentdetects hydrolysis mediated by an esterase present in eukaryotic but notbacterial cells. Poor E-selectin stimulation by H. pylori and P.gingivalis could not be attributed to endothelial cell toxicity sinceeven high concentrations of these bacteria were not toxic as assayed bythese parameters. IL-1β was added as an additional control of theability of endothelial cells to express E-selectin in the presence ofbacteria. At IL-1β concentrations ranging from 0.03 to 20 ng/ml, 10% P.gingivalis whole cells did not affect the ability of the endothelialcell monolayer to express E-selectin. In contrast, calcein-AM hydrolysisassays revealed that concentrations of E. coli of 10⁸ cfu/ml and greaterwere toxic.

The potential of isolated LPS preparations to directly stimulateE-selectin expression was also examined. Similar experiments are alsodescribed in detail in Example III below. As shown in FIG. 3, serum wasrequired to obtain a significant E-selectin response to E. coli LPS. Theresponse to E. coli LPS was potent, with as little as 1 ng yieldingsignificant expression. In contrast, but similar to the data obtainedwith whole cells, LPS obtained from P. gingivalis and H. pylori did notinduce E-selectin expression. Also, similar to what was observed withwhole cells, P. aeruginosa required significantly more LPS to obtain anequivalent E. coli level of E-selectin expression. In addition to thedata presented in FIG. 3, LPS obtained from three additional strains ofP. gingivalis (A7A1-28, A7436 and 5083) and LPS obtained from B.forsythus also failed to stimulate E-selectin expression (a minimum ofthree separate experiments at 1 ng/ml was performed with each LPS). Nostimulation of E-selectin was observed when these LPS preparations wereexamined with or without the addition of human serum. Cell wallsobtained from P. aeruginosa ATCC 27316 yielded a similar significantlyreduced response. Cell walls obtained from H. pylori ATCC 43504 or P.gingivalis ATCC 33277 also were unable to elicit E-selectin expression(FIG. 4). Calcein-AM hydrolysis assays confirmed that these preparationswere not toxic to the endothelial cells during the assay.

EXAMPLE III

The ability of P gingivalis LPS to stimulate E-selectin expression wasexamined in this series of experiments. In addition, the effect of LPSobtained from a related organism, Bacteroides forsythias, also believedto be associated with adult periodontal disease was examined.

The effect of various LPS preparations on E-selectin expression wasdetermined by adding LPS preparations to HUVEC monolayers atconcentrations of 0.0001, 0.001, 0.01, 0.1 and 1.0 μg/ml, as describedin Example I above. LPS from E. coli was obtained from Sigma (0111:B4);LPS was purified from P. gingivalis 33277 and A7A1-28 by the phenolwater method of Westphal and Jann, in Methods in Carbohydrate Chemistry5: 83-91, R. L. Whistler, ed., Academic Press, Inc., New York; LPS waspurified from B. forsythias and P. gingivalis strain 5083 by the coldMg/ETOH procedure (Darveau and Hancock, J. Bacteriol, 155:831-838(1983)). All LPS preparations were suspended in dH₂ 0. LPS preparationswere determined to be free from contaminating nucleic acid and proteinand subjected to gas chromatographic analysis for sugar and fatty acidcomposition. The composition of the LPS preparations was consistent withpreviously reported characterizations. Each assay was performed on threeseparate occasions in duplicate.

The results from typical assays are depicted in FIG. 5. No stimulationof E-selectin was observed with these LPS preparations at concentrations1000 fold greater than that needed to obtain a significant signal fromE. coli LPS.

As the presence of serum has been shown to be necessary for LPS inducedE-selectin expression (Frey et al., J. Exp, Med,. 176:1665 (1992); Puginet al., Proc. Natl. Acad. Sci. USA 90:2744 (1993)), the possibility thatserum may have interfered with the presentation of the LPS to theendothelial cell was examined. E-selectin stimulations were conducted inthe absence of human serum and with serum which had been heatinactivated at 56° C. for 30 minutes. In the absence of serum or inheat-inactivated serum, 10,000 fold more E. coli LPS was needed toobtain a significant E-selectin response when compared to stimulationwith NHS. Once again, however, no E-selectin response to P. gingivalisLPS was observed.

EXAMPLE IV

Having demonstrated that LPS from P. gingivalis was unable to stimulateE-selectin upregulation, the ability of P. gingivalis LPS to block theupregulation of E-selectin that had been stimulated with either E. coliLPS or TNF was also determined.

Preparations of LPS from E. coli and P. gingivalis were mixed and thenadded to endothelial cells. P. gingivalis LPS, obtained from strain ATCC33277 as described in Example III, was mixed at varying ratios with twopreparations of E. coli LPS or TNF as indicated in FIG. 6A or FIG. 6B,respectively, prior to addition to endothelial cells. Assay ofE-selectin was performed as described in Example I. Three separateexperiments were performed with similar results. The result of a typicalexperiment are depicted.

As shown in FIG. 6A, ratios of P. gingivalis LPS that were 10 to 100fold higher than the E. coli LPS were able to significantly block thestimulation of E-selectin. As shown in FIG. 6B, when similar mixingexperiments were performed with P. gingivalis LPS and tumor necrosisfactor, no inhibition of E-selectin expression was observed.

Studies were then performed in an effort to determine which portion ofthe P. gingivalis LPS was responsible for the ability to selectivelyinhibit E-selectin expression. P. gingivalis LPS was selectivelydegraded into its lipid A and polysaccharide components (LPS-PS) byhydrolysis in the presence of 1% acetic acid for 30 min. Fractions wereseparated by centrifugation and analyzed for their lipid andcarbohydrate content by gas chromatography. The composition of eachpreparation was consistent with previously reported data. E. coli LPS(10 ng/ml) was added to an endothelial cell monolayer as described inFIG. 1 (control); the same amount of E. coli LPS was mixed with 10 μg/mlof either P. gingivalis LPS, LPS-PS, or Lipid-A and analyzed forE-selectin expression.

FIG. 7 shows the results of the E-selectin expression inhibition studiesby the P. gingivalis LPS fractions. In three separate experiment neitherisolated lipid A nor the polysaccharide component when added at 10 μg/mlwere able to block E-selectin activation by 10 ng/ml E. coli LPS. Theinability of these fractions to block when added at 100 fold excesssuggests that both components of the LPS molecule may be required forinhibition of E-selectin expression. However, selective degradation of akey LPS component due to the hydrolysis procedure was not ruled out.

P. gingivalis LPS also blocked E-selectin expression by Actinobacillusactinomycetemcomitans and Salmonella typhimurium LPS.

In other experiments P. gingivalis LPS was shown to block E-selectinexpression stimulated by cell walls obtained from Leptotrichia buccalis(ATCC 14201), E. coli ATCC 29552, Haemophilus parainfluenzae (BMS C128),Neisseria flavescens (ATCC 13120), Eikenella corrodens (ATCC 23834), andFusobacterium nucleatum (ATCC 25586).

EXAMPLE V

Antibodies that bind to P. gingivalis were examined for the ability toprevent or reverse the inhibition of E-selectin expression that ismediated by P. gingivalis LPS.

Endothelial cells were stimulated with 10 ng/ml of E. coli LPS. As shownin FIG. 8, last column, there was an E-selectin response when no P.gingivalis LPS was mixed with the E. coli LPS prior to addition to theendothelial cells. If however, 0.5 μg/ml P. gingivalis LPS was pre-mixedwith the E. coli LPS prior to addition to the endothelial cells, therewas a complete inhibition of the E-selection response, as shown in thefirst column of FIG. 8 labeled "P.g. LPS".

Varying dilutions of pre-immune and immune rabbit sera (immunized withwhole P. gingivalis) were then preincubated with the E. coli and P.gingivalis LPS preparations. Preimmune and immune rabbit sera werediluted 1/12.5, then serially for several two-fold dilutions in"Endochow" (M-199, GIBCO with 4 mM L-glutamine, 90 μg/ml heparin, 1 mMsodium pyruvate) without serum or endothelial cell growth factors. Thediluted sera was combined 1:1 with P. gingivalis LPS (concentration at 1μg/ml) in the same media and the mixture left at room temp. for about 1hr. 100 μl of the rabbit sera/P. gingivalis LPS mixture was then mixedwith 100 μl of E. coli 0111:B4 LPS (final concentration of 10 ng/ml).100 μl of this mixture was then added to HUVECs (previously washed 1×with media, without serum) to stimulate E-selectin expression at 37° C.under 5% CO₂ for 4 h. After stimulation the media was removed from theplates and the cells washed 2× in cold PBS (100 μl per well). 100 μl of0.5% glutaraldehyde (in cold PBS) was added per well and plates cooledto 4° C. for 10 minutes. Plates were then washed 4× using PBS with 3%normal goat serum and 0.02M EDTA (200-300 μl per well). After the lastwash, 200-300 PBS/goat serum/EDTA was added per well and the platesstored overnight at 4° C. All antibodies were diluted in PBS/goatserum/EDTA described above. The blocking reagent was then removed fromthe wells and 100 μl of primary antibody added (usually a mouseanti-ELAM monoclonal, at 0.25 μg/ml). Plates were incubated at 37° C.for 1 hr and then washed 4× in PBS/goat serum/EDTA (200-300 μl perwell), and 100 μl/well of second step antibody (Jackson Labs F(ab')₂goat anti-mouse IgG, Fc specific, HRP conjugated #115-036-071!) was thenadded. Plates were incubated at 37° C. for 1 hr, washed 4× with PBS/goatserum/EDTA, and received 100 μl of chromogen reagent per well (TMBdiluted in substrate buffer). After color development for about 20 min.the reaction was stopped with 100 μl of 1N H₂ SO₄ per well and theplates were read at 450/630

As shown in FIG. 8, as the concentration of anti-P. gingivalis seraincreased there was an increase in the E-selection signal compared topre-immune sera. This indicated that the anti-P. gingivalis antibodiescould inhibit the ability of P. gingivalis LPS to block E. coli LPSmediated E-selectin expression.

EXAMPLE VI P. gingivalis and H. pylori Do Not Promote NeutrophilAdhesion to Endothelial Cells

This Example demonstrates that in contrast to E. coli, P. gingivalisdoes not induce human endothelial cells to be adhesive to neutrophils.The Example also demonstrates that P. aeruginosa was a very poor inducerof neutrophil adhesion, and H. pylori LPS did not induce neutrophiladhesion to endothelium.

For human neutrophil preparation, blood was obtained from normal healthyhuman volunteers by venipuncture using heparin containing syringes.Neutrophils were isolated using density gradient centrifugation withPolymorphprep™ (Nycomed Pharma AS, Oslo, Norway) as described by themanufacturer. Contaminating red blood cells were lysed as described inMagnuson et al., J. Immunol, 143:3025-3030 (1989), which is incorporatedherein by reference. A portion of the neutrophil preparation wasstained, checked for purity, and the remaining cells were suspended to4×10⁶ cells/ml for fluorescent labeling. Neutrophils were labeled withBCECF-AM (Molecular Probes, Inc., Eugene, Oreg.) according tomanufacturer's instructions. Specifically, neutrophils were incubatedwith 10 mM BCECF-AM in DMSO for 15 min. in the dark, an equal volume ofRPMI containing 5% FBS was added and the cells were centrifuged.Neutrophils were washed in PBS and then suspended at 2×10⁶ cells/ml inRPMI containing 1% FCS and kept in the dark.

For the neutrophil adhesion assay the basic procedure described byMagnuson et al., supra, was followed. HUVEC monolayers were prepared asdescribed above for the E-selectin expression assay except that 4×10⁴HUVEC/well were added to a 96 well plate. HUVEC monolayers werestimulated for 4 hrs with LPS or cell wall preparations as describedabove for the E-selectin expression assay. After the 4 hr stimulationthe HUVEC monolayers were washed with PBS containing 5% FCS and labeledneutrophils were added (0.1 ml of the stock solution, representing about2×10⁵ cells/well). The neutrophil/HUVEC cell preparation was coveredwith foil and placed on a shaker with mild agitation for 30 min atambient temperature. After 30 min the nonadherent neutrophils wereremoved by careful aspiration, followed by two washes with PBScontaining 5% FCS. After washing, 0.1 ml of a solution containing 50 mMTris pH 8 and 1% SDS was added to the HUVEC monolayers and the plate wasread on a Fluorescence Concentration Analyzer (Baxter ScientificProducts, Philadelphia, Pa.) with excitation at 485 nm and emission at535 nm. The percent of total neutrophils which adhered in each assay wasdetermined by constructing a standard curve with varying amounts oflysed neutrophils plotted against fluorescent intensity. Routinely, atnear maximum binding (20,000 units) approximately 50% of the neutrophilswere bound.

The results showed that although E. coli was a potent inducer ofneutrophil adhesion, no adhesion was detected when P. gingivalis or H.pylori LPS were examined and significantly lower neutrophil adhesionoccurred after endothelial cell exposure to P. aeruginosa LPS (FIG. 9).

Examination of neutrophil adhesion as opposed to E-selectin expressionallowed a determination whether bacterial preparations obtained from P.gingivalis or H. pylori would promote neutrophil adhesion by E-selectinindependent mechanisms. The lack of neutrophil adhesion demonstratesthat these organisms could not induce endothelial cell adhesiveness byE-selectin independent mechanisms.

EXAMPLE VII P. gingivalis Inhibits E. coli-LPS Induction of MonocyteChemoattractant Protein from Human Gingival Fibroblasts

This Example demonstrates that P. gingivalis LPS does not stimulate theproduction of monocyte chemoattractant protein (MCP-1) from humangingival fibroblasts. MCP-1 is a chemokine, synthesized in response toan inflammatory stimulus and believed to attract leukocytes to the siteof inflammation. MCP-1 has been shown to be expressed in normalperiodontal tissue and is believed to play a role in the protection ofhost tissue from damage incurred by the presence of neutrophils.

In these experiments 10 ng/ml of E. coli LPS was a potent inducer ofMCP-1 mRNA whereas 1 μg of P. gingivalis LPS did not result in MCP-1mRNA expression. In addition, 50 ng/ml of P. gingivalis LPS was able toblock E. coli induced MCP-1 expression. These data extend theobservations with P. gingivalis LPS to include an inhibitory effect onhuman gingival fibroblasts and chemokine expression.

Primary human gingival fibroblasts (HGF-60) cells were grown in DMEMmedia supplemented with 10% FBS, sodium pyruvate, glutamine, penicillinand streptomycin. Early passage cultured cells were plated at 1 to 2×10⁶cells per 100 mm culture dish and treated with various concentrations oflipopolysaccharides (LPSs) isolated from either E. coli (10 ng/ml E.coli LPS 011:B4 (Sigma)) or P. gingivalis (1000 ng/ml P. gingivalis LPS)or mixture of both LPSs, in 2% FCS for 18 to 24 hours. The cells weresubsequently harvested for RNA isolation.

Total RNA was isolated by a single step guanidiniumthiocyanate-phenol-chloroform method of Chomczynski and Sacchi, Anal.Biochem. 162:156-159 (1987), incorporated herein by reference. Cellswere lysed directly on the dish using RNAStat-60 solution, (Tel-Test"B", Inc., Friendswood, Tex.) and the RNA was enriched for mRNA bycolumn purification using RNAStat-30 kit (Tel-Test "B", Inc.) usingmanufacturer's protocol.

RNA samples (15 μg/lane) were run on 1.0% vertical slab agarose gelcontaining 6% formaldehyde in 1×MOPS buffer. Gels were run at 70-75Volts for 4 hours and electrophoresed RNA was transferred to Hybond-Nmembranes (Amersham Life Sciences) in 20×SSC overnight. Blots were thencrosslinked using a UV Stratalinker (Stratagene) and were prehybridizedfor 4 hours at 42° C. in buffer containing 50% formamide, 4×Denhardts,5×SSC, 1% SDS, 10 mM Tris-HCl pH 7.5, 50 μg/ml salmon sperm DNA.

Northern blots were hybridized using ³² P! labeled probe for MCP-1. Theprobe was obtained by random primed labelling using ³² P! dCTP by MCP-1encoding cDNA fragment. Hybridizations were performed at 42° C.overnight in prehybridization buffer containing 1 to 2×10⁶ cpm ³² Plabelled probe per ml. Blots were subsequently washed in 0.2×SSC, 0.1%SDS at 65° C., and either autoradiographed or scanned on MolecularDynamics Phosphorimager. Images were subsequently quantitated.

The results, shown in FIG. 10, demonstrated that E. coli LPS produced anintense signal when incubated without P. gingivalis LPS. No signal wasobserved, however, when cells were incubated with P. gingivalis LPS(1000 ng/ml). Co-incubation of E. coli LPS and 50 ng/ml P. gingivalisLPS resulted in the almost complete inhibition of the E. coli LPSmediated expression of MCP-1 RNA.

EXAMPLE VIII P gingivalis Inhibits K Light Chain Expression

This Example demonstrates that the inhibitory effect of P. gingivalisLPS includes immunoglobulin producing cells, suggesting that theimmunosuppressive effect of such organisms may have wide implications.

The mouse cell line 70 Z/3 is a pre B lymphoma cell line which is frozenin immunoglobulin expression at an early stage, thus it hasconstitutively expressed μ heavy chain on its surface but does notcontain κ light chain. Miller et al., Mol. Cell. Biol. 11:4885-4894(1991), incorporated herein by reference. κ light chain can be inducedto be expressed by E. coli LPS.

In this experiment 70 Z/3 cells were incubated with the concentrationsof E. coli LPS indicated on the X axis were co-incubated with 10 μg/mlof P. gingivalis LPS. After 18 hrs incubation the expression of κ lightchain on the cell surface was examined by fluorescent activated cellsorting (FACS). The results are presented as mean fluorescence.

P. gingivalis LPS did not stimulate, but rather, inhibited the abilityof E. coli LPS to stimulate the expression of κ light chain in mouse 70Z/3 cells. As shown in FIG. 11, although E. coli LPS at 0.1 ng/ml wasable to stimulate expression of κ light chain as measured byimmunofluorescence, 10 μg/ml of P. gingivalis LPS did not result in κlight chain expression. In addition, P. gingivalis LPS was able to blockE. coli induced κ light chain expression when LPS sample were pre-mixedbefore addition to the cells. These data thus extend the inhibitoryeffect of P. gingivalis LPS to include immunoglobulin producing cells.

EXAMPLE IX Monoclonal Antibodies Block P. gingivalis Mediated Inhibitionof E-Selectin and Neutrophil Adhesion

This Example demonstrates that monoclonal antibodies directed against P.gingivalis LPS can restore near normal E-selectin expression on humanendothelial cells exposed to E. coli and P. gingivalis LPS.

These experiments were performed essentially as described in Example Vabove, except that monoclonal antibody 7F12 which binds P. gingivalisLPS and monoclonal antibody ACE12-3B4 which does not bind P. gingivalisLPS were examined. In this experiment, 0.7 ug/ml P. gingivalis LPS or3.3 ng/ml E. coli 011B:4 LPS was added to endothelial cells, eitherindividually or in combination. In addition, monoclonal antibodyACE12-3B4 (which binds to E. coli LPS) or monoclonal antibody 7F12 wereadded to the combination of LPS preparations. Mouse polyclonal sera(from a mouse immunized with P. gingivalis LPS) and the prebleed serawere also added to the combination of LPS preparations.

The results showed that when P. gingivalis LPS was added to theendothelial cells, little or no stimulation was observed. When E. coliLPS was added a significant E-selectin response was obtained. When acombination of P. gingivalis LPS and E. coli LPS were added the ELISAsignal was reduced to near background (0.2). A slight increase in thesignal was obtained if a negative control antibody (ACE12-3B4) or mousepolyclonal pre-bleed sera was added to the combination. A furtherincrease in the signal was obtained if a monoclonal antibody to P.gingivalis LPA (7F12) or mouse polyclonal sera generated by immunizationwith P. gingivalis LPS was added to the combination. These datademonstrate that antibodies to P. gingivalis LPS can inhibit the abilityof this LPS to block E-selectin expression.

The ability of the monoclonal antibody to P. gingivalis LPS to inhibitthe P. gingivalis-mediated inhibition of neutrophil binding was thendetermined. This experiment was performed essentially as described abovein Example VI. The neutrophil binding assays were performed with theaddition of monoclonal antibodies (purified monoclonal antibodies wereadded at 10 ug/ml). Monoclonal antibody 4B2 does not bind P. gingivalisLPS (it binds to P. aeruginosa LPS), monoclonal antibodies 7F12, 6E12,and 5B9 all bind P. gingivalis LPS. In this experiment 1 ug/ml P.gingivalis LPS was added to endothelial cells or 10 ng/ml E. coli0111B:4 LPS, either individually or in combination lanes. In addition,monoclonal antibody 4B2 or monoclonal antibodies 7F12, 6E12, and 5B9were added to the combination of LPS preparations.

The results showed that P. gingivalis LPS did not promote the adhesionof neutrophils to endothelial cells. In contrast, E. coli LPS was ableto significantly increase the ability of endothelial cells to bind humanneutrophils. When a combination of P. gingivalis and E. coli LPS wasadded there was a significant reduction in the ability of theendothelial cells to bind human neutrophils, confirming the P.gingivalis LPS can block the ability of E. coli LPS to promoteneutrophil binding. Further, H. pylori LPS is similar to P. gingivalisin that it inhibited E. coli LPS-mediated stimulation of neutrophiladhesion. When a negative control antibody was added to the P.gingivalis and E. coli LPS combination no effect on neutrophil bindingwas observed. In contrast, when monoclonal antibodies to P. gingivalisLPS were added to the P. gingivalis and E. coli LPS combination asignificant increase in neutrophil binding was observed. This datademonstrates that monoclonal antibodies to P. gingivalis LPS can blockthe P. gingivalis LPS inhibition of neutrophil binding.

EXAMPLE X P. gingivalis Does Not Induce Acute Inflammation In vivo

This Example demonstrates that P. gingivalis does not generate an acuteinflammatory response in vivo, in contrast to the acute responseresulting from injections of E. coli LPS or organisms.

Acute inflammation is characterized by margination of leukocytes in thevasculature and their migration into tissues at the reaction site. Thisprocess of leukocyte trafficking involves not only activated leukocytesbut activated endothelial cells as well. A mouse model of acuteinflammation was established to examine the cellular and temporalpattern of mRNAs expressed for various chemokines and cell adhesionmolecules.

In these studies, mice were injected intramuscularly with 0.2 mg of E.coli lipopolysaccharide (LPS), a potent inflammatory mediator, and thensacrificed either at 4 or 24 hours. The injected muscles were excisedcryosectioned and prepared for in situ hybridization to detectexpression of monocyte chemoattractant protein 1 (MCP-1) mRNA, a memberof the C-C or β-subfamily of chemokines, for E- and P- selectin mRNAs,the cell adhesion molecules induced on endothelial cells which arecritical for leukocyte binding to endothelium and subsequent migrationto extravascular tissue sites, or for fibroblast inflammatory chemokine(FIC).

Balb/c mice were injected in the gastrocnemius muscle with 0.2 mg of E.coli LPS (0111:B4; Sigma). Control mice received saline. For studiesusing bacteria, E. coli (D471 strain) and Porphyromonas gingivalis(strain 33277 P. gingivalis) were grown on trypticase agar platesovernight and Brucella blood agar plates anaerobically for five days,respectively. Plated bacteria were suspended to predeterminedconcentrations and animals were injected with either 10², 10⁵, 10⁷ E.Coli or 10⁶, 10⁸, 10¹⁰ P. gingivalis. The number of cells injected intoeach animal was confirmed by viable colony counts performed by standardmethods.

Mice were sacrificed at either 4 or 24 hours and the muscles excised.For frozen sections, muscles were embedded in OCT compound andcryosectioned. For paraffin sections, muscles were first fixed in 4%paraformaldehyde and then embedded and sectioned.

Probes for in situ hybridization were either ribonucleic acid probesgenerated from cDNAs or oligonucleotide probes designed from known cDNAsequences. The MCP-1 riboprobe was made from a mouse MCP-1 cDNA in a TAPCR vector (Invitrogen). Template was prepared by PCR directly from theplasmid using Universal and M13 reverse primers. After phenol/chloroformextraction and isopropanol precipitation, the 250 bp fragment was usedas a template for transcription by Sp6 RNA polymerase to generate anantisense probe. The IL-1 beta cDNA was obtained from Hoffman-LaRoche,Nutley, N.J. For oligonucleotide probes, a cocktail of probes were used.In the case of E-selectin, two oligos were used: one to the lectinbinding domain and another to the EGF region. Oligos for P-selectin werealso made to these regions in addition to one of the firm complementdomain. Three oligo probes were also made to the coding regions of FICand MCP-1. Paraffin and frozen sections were prepared for in situhybridization as described in Sandell et al., J. Cell Biol.114:1307-1319 (1991), and Aigner et al., Virch. Archiv. B Cell Pathol.62:337-345 (1992), incorporated herein by reference.

The results for selectin mRNA expression in muscle which had beeninjected with E. coli LPS were as follows. In situ hybridization ofserial sections using oligonucleotide probes (a cocktail of two or threenon-overlapping radiolabeled oligonucleotide probes to either E- orP-selectin were used) showed expression of E- and P- selectin mRNAs inendothelial cells from frozen sections of muscle taken 4 hours after theinjection of E. coli LPS (0.2 mg). Thus, 4 hrs after the induction ofinflammation, E- and P-selectin mRNAs were strongly expressed inendothelial cells of capillaries in the endomysium and larger vessels ofthe perimysium. Strong hybridization for selectin mRNAs was similarlyobserved in sections of 24 hr inflamed muscle. Muscle injected with PBSas a control showed positive cells only along a narrow tract of tissueprobably representing inflammation caused by the insertion of theneedle.

The in situ hybridization using a ribonucleic acid probe also showedexpression of monocyte chemotactic protein-1 (MCP-1) mRNA ininflammatory cells from frozen sections of muscle taken 4 hours afterthe injection of E. coli LPS (0.2 mg). Frozen section of muscle taken 4hours after the injection of PBS showed the absence of MCP-1 mRNA andserved as a control. In situ hybridization using a cocktail of 3oligonucleotide probes gave an identical pattern of hybridization. Thus,after 4 hrs, monocyte chemoattractant protein 1 (MCP-1) mRNA wasdetected in leukocytes using specific radiolabeled ribonucleic acidprobe. In these samples, mononuclear but not polymorphonuclear cells,expressing MCP-1 mRNA were abundant throughout the tissue section in theconnective tissue septa between muscle bundles (pertmysium) and betweenindividual fibers (endomysium). In contrast, after 24 hrs the number ofMCP-1 mRNA expressing cells appeared to abate.

This animal model of acute inflammation was then used to compare theaction of E. coli with that resulting from injections of P. gingivalis.In situ hybridization in muscle tissue using ribonucleic acid probeshowed expression of monocyte chemotactic protein-1 (MCP-1) mRNA in aparaffin section of muscle taken 4 hours after the injection of 10⁷ E.coli. High magnification showed the expression of MCP-1 mRNA inmonocytes but not PMNs. The expression of MCP-1 was observed in only afew cells in a frozen section of muscle taken 4 hours after theinjection of 10¹⁰ P. gingivalis.

The mRNA expression of IL-1 in E. coli and P. gingivalis-injected musclewas also detected by in situ hybridization using ribonucleic acid probe.Expression of IL-1 mRNA was detected in a frozen section of muscle taken4 hrs after the injection of 10⁵ E. coli, but the expression of IL-1 wasabsent in a frozen section of muscle taken 4 hrs after the injection of10⁸ P. gingivalis.

mRNA expression of fibroblast inflammatory chemokine (FIC) was alsodetermined in E. coli- and P. gingivalis-injected muscle. In situhybridization using oligonucleotide probes showed expression of FIC mRNAin frozen sections of muscle taken 4 hrs after the injection of 10⁵ E.coli, whereas the expression of FIC was absent in frozen sections ofmuscle taken 4 hrs after the injection of 10⁸ P. gingivalis.

By using this system it was thus demonstrated that monocytes fromsections of muscle 4 hrs after an injection of E. coli expressed MCP-1mRNA whereas relatively few cells from muscles injected with even higherdoses of P. gingivalis expressed this mRNA. Similarly, inflammatorycells from E. coli injected muscle also expressed IL-1 and fibroblastinflammatory chemokine (FIC) while those from P. gingivalis did not.These data confirmed that E. coli induced acute inflammation in theanimal but P. gingivalis did not. These results are consistent with thein vitro observations and suggests that P. gingivalis has developed acell wall and LPS composition that is poorly recognized by the mammalianinflammatory system.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for screening compounds which inhibitthe ability of P. gingivalis lipopolysaccharide to inhibit theextravasation of leukocytes from the vascular endothelium to gingivaltissues, comprisingcontacting cells which are capable of expressing aselectin molecule with P. gingivalis lipopolysaccharide in the presenceand absence of the compound being screened for the ability to inhibit P.gingivalis lipopolysaccharide-induced inhibition of selectin expression;stimulating expression of the selectin molecule by the cells; andmeasuring the expression of the selectin molecule by the cells in thepresence or absence of said compound and therefrom determining theability of said compound to inhibit P. gingivalislipopolysaccharide-induced inhibition of selectin expression.
 2. Themethod of claim 1, wherein the cells capable of expressing selectinmolecules are human umbilical vein endothelial cells.
 3. The method ofclaim 1, wherein the expression of the selectin is induced by E. colilipopolysaccharide, tumor necrosis factor, or interleukin-1.
 4. Themethod of claim 1, wherein the selectin molecule is an E-selectin. 5.The method of claim 1, wherein the selectin molecule is a P-selectin. 6.The method of claim 1, wherein the cells capable of expressing selectinmolecules are endothelial cells.